Introducing IsoMad, a compilation of isotopic datasets for Madagascar

We present the first open-access, island-wide isotopic database (IsoMad) for modern biologically relevant materials collected on Madagascar within the past 150 years from both terrestrial and nearshore marine environments. Isotopic research on the island has increasingly helped with biological studies of endemic organisms, including evaluating foraging niches and investigating factors that affect the spatial distribution and abundance of species. The IsoMad database should facilitate future work by making it easy for researchers to access existing data (even for those who are relatively unfamiliar with the literature) and identify both research gaps and opportunities for using various isotope systems to answer research questions. We also hope that this database will encourage full data reporting in future publications.

At UCSC, approximately 0.7 mg of fur was analyzed on a Finnigan Thermo Electron Delta+XP continuous flow system (Bremen, Germany) connected to a Carlo Erba elemental analyzer (Milan, Italy) via a Conflo III interface (Valencia California, USA.Two reference materials were analyzed along with the samples: homogenized gelatin (PUGEL) and International Atomic Energy Agency (IAEA) acetanilide.Various masses of PUGEL were used to account for sample size (linearity) at the beginning of the run, and additional PUGEL replicates were used to correct for drift throughout each run.Analytical precision was monitored using acetanilide.Based on other work in the lab [1][2][3], precision (±1 SD) is estimated to have been <0.2‰ for   C was and <0.1‰ for   N.
At UC, approximately 0.45 mg of fur was analyzed on a Costech Elemental Analyzer connected to a Thermo Scientific Delta V IRMS (Bremen, Germany) via a Costech Conflo IV interface (Valencia California, USA).Four reference materials were analyzed along with the samples: Powdered caffeine; US Geological Survey (USGS) 41; PUGEL; and powdered glycine.Data were corrected for linearity and drift using powdered caffeine (various masses analyzed at the beginning of the run and then additional replicates with masses more similar to samples were interspersed throughout the run).We accounted for scale using caffeine and USGS41 (n=4 total) analyzed at the beginning and end of the run.Analytical accuracy was evaluated using PUGEL (n=3) and glycine (n=3) that were interspersed across each run.Precision was monitored using all four reference materials.Based on other work in the lab [1][2][3], accuracy and precision are estimated to have been ca.0.3‰ for carbon and <0.1‰ for nitrogen.
Dried bone samples were processed following Crowley et al. [4].Samples were soaked in 0.5N HCl at 4°C until gummy.They were rinsed 5x with ultrapure water and then repeatedly sonicated in petroleum ether until all visible lipids were removed.They were again rinsed 5x with ultrapure water and freeze dried.Samples were analyzed at the Stable Isotope Biogeochemistry Facility at the University of Cincinnati in Cincinnati Ohio (UC).Approximately 0.4 mg of each pre-treated and dried bone were weighed into a tin boat and samples were then combusted on a Costech Elemental Analyzer connected to a Thermo Scientific Delta V IRMS (Bremen, Germany) via a Costech Conflo IV interface (Valencia California, USA).Four reference materials were analyzed along with the samples: Caffeine; US Geological Survey (USGS) 41; PUGEL; and glycine.Data were corrected for linearity and drift using powdered caffeine (various masses analyzed at the beginning of the run and then additional replicates with masses more similar to samples were interspersed throughout the run).We accounted for scale using caffeine and USGS41 (n=4 total) analyzed at the beginning and end of the run.Analytical accuracy was evaluated using PUGEL (n=3) and glycine (n=3) that were interspersed across the run.Precision was monitored using all four reference materials.Based on historic performance of the lab, accuracy and precision across runs were <0.1‰ for both isotopes.
Pretreatment Code: 4 Site: Beza Mahafaly Contributors: James Loudon, Matt Sponheimer Plant samples were collected at the Beza Mahafaly Special Reserve (23º30'S latitude, 44 º 40'E longitude), located in southwest Madagascar.Plant samples were placed in labeled bags that included, location of collection, plant organ, plant species name, and local Malagasy plant name.Each sample was wiped clean of any dust or particulate.Plant samples were desiccated fully at the field site in a camp oven.All samples were transported to the University of Colorado, Boulder and further dried in a benchtop oven at 40°C for 24 hours.All plant samples were ground into a fine powder using a mortar and pestle.Approximately 2.5 mg of plant sample powder was placed in tin capsules.Tin capsules were combusted in an elemental analyzer (Carlo-Erba, Milan, Italy) and analyzed for stable carbon and nitrogen isotope abundances using a flow-through inlet system on a continuous flow isotope ratio mass spectrometer (Finnigan, Bremen, Germany). 13C/ 12 C and 15 N/ 14 N ratios are expressed in delta (δ) notation in parts per thousand or per mil (‰) relative to the Vienna Peedee Belemnite and atmospheric N2 standards.All samples for this study were analyzed during one analytical run for which standard deviations of replicate measurements of internal standards (protein and yeast) were <0.1‰ for both δ 13 C and δ 15 N values.

Pretreatment Code: 5 Site: Toliara Contributor: Loïc Michel
As soon as possible after collection, animals were dissected to separate soft and nonmetabolically active tissues (e.g.muscle, tegument) or, when body size was small, were used whole [5].Obtained samples were oven-dried at 50°C for 72 hours.They were subsequently ground to a homogeneous powder using mortar and pestle or a mixer mill (MM301, Retsch, Haan, Germany) depending on toughness.Samples containing hard inorganic carbon parts that could not be physically removed were acidified by exposing them to HCl vapors for 48h in an airtight container [6].Stable isotope ratio measurements were performed via continuous flow -elemental analysis -isotope ratio mass spectrometry (CF-EA-IRMS) at University of Liège (Belgium), using a vario MICRO cube C-N-S elemental analyzer (Elementar Analysensysteme GMBH, Hanau, Germany) coupled to either an IsoPrime100 or a precisION isotope ratio mass spectrometer (Isoprime, Cheadle, United Kingdom).Isotopic ratios were expressed using the widespread δ notation (Coplen 2011), in ‰ and relative to the international references Vienna Pee Dee Belemnite (for carbon), atmospheric air (for nitrogen) and Vienna Canyon Diablo Troilite (for sulfur).IAEA (International Atomic Energy Agency, Vienna, Austria) certified reference materials sucrose (IAEA-C-6; δ 13 C = -10.8± 0.5‰; mean ± SD), ammonium sulfate (IAEA-N-1; δ 15 N = 0.4 ± 0.1‰; mean ± SD ; or IAEA-N-2; δ 15 N = 20.4 ± 0.1‰; mean ± SD) and silver sulfide (IAEA-S-1; δ 34 S = -0.3‰; or IAEA-S-1; δ 34 S = 22.6‰) were used as primary analytical standards.Sulfanilic acid (Sigma-Aldrich; δ 13 C = -25.6 ± 0.4‰; δ 15 N = -0.13± 0.4‰; δ 34 S = 5.9 ± 0.5‰; means ± SD) was used as secondary analytical standard.Standard deviations on multi-batch replicate measurements of secondary and internal lab standards (amphipod crustacean muscle, seabass muscle or seagrass leaves, according to sample nature) analyzed interspersed with samples (one replicate of each standard every 15 analyses) were typically 0.2‰ for both δ 13 C and δ 15 N and 0.4‰ for δ 34  Two grams of dried plant biomass were ground in a Retsch MM200 mill (Retsch GmbH, Hann, Germany) and subsequently stored dry in 2 mL Eppendorf tubes until further analysis.Samples of around 2 mg were weighed into tin cups (HEKAtech, Germany) for analysis in a stable isotope ratio mass spectrometer (nu Horizon, Nu Instruments Limited, UK) linked to an elemental analyzer (EURO-EA 3000, Euro Vector, Italy) in continuous flow configuration (set-up by HEKAtech, Germany).
Pretreatment Code: 8 Site: Toliara Contributor: Lucas Terrana, Gilles Lepoint, Richard Rasolofonirina, Igor Eeckhaut Analytical procedure: Fresh mangrove crabs were measured on their cephalothorax, then sexed, and dissected to sample the left cheliped (i.e.first thoracic limb of crabs bearing a pincer or claw or nipper).Internal muscle of the cheliped were extracted, taking attention not to sample cuticle fragments which are carbonated.Samples were dried at 60°C during 48h and then ground using pestle and mortar.Mangrove leaves were dried at 60°C during 48h and then ground using a Retsch MM 301 micro-ball mill (2 min, 25 Hz) to obtain a homogeneous powder, which was weighed and placed into tin capsules.Stable isotope ratio measurements were performed via continuous flow -elemental analysis -isotope ratio mass spectrometry (CF-EA-IRMS) at University of Liège (Belgium), using a vario MICRO cube C-N-S elemental analyzer (Elementar Analysensysteme GMBH, Hanau, Germany) coupled to either an IsoPrime100 isotope ratio mass spectrometer (Isoprime, Cheadle, United Kingdom).
Traceability: Isotopic ratios are presented as δ values (‰), expressed relative to the vPDB (Vienna Peedee Belemnite) standard and to atmospheric N2 for carbon and nitrogen, respectively.Reference materials were IAEA-N1 (δ 15 N=+0.4 ± 0.2‰) and IAEA CH-6 (sucrose) δ 13 C= -10.4 ± 0.2‰).Each reference material was measured in duplicate at the beginning and end of each analytical batch.Uncertainty: Experimental precision (based on the standard deviation of replicates of an internal standard) was 0.3 and 0.4‰ for carbon and nitrogen, respectively.Usage Notes S1.

Example 1:
Figure S1.Illustration of δ 13 C and δ 15 N values from the tissues of selected primary producers (squares) and consumers (circles) collected from the reef flats surrounding Toliara (n=146).Consumer tissues are muscle, with the exception of tegument analyzed from Synapta maculata, and colored large points and lines mark group means and standard deviations.Note that dietary inference through stable isotope analysis of consumer tissues relies necessarily on the distinct isotopic composition of different dietary sources, and IsoMad makes it easy to explore diverse sources.This example highlights the distinct δ 13 C values of primary producer tissues and a range of trophic levels reflected in consumer tissue δ 15 N values [10][11][12].Some of these groups may provide useful endmembers for future work involving dietary inference from stable isotope data.
For example, as expected, the observed negative relationship between water availability and plant δ 15 N values is stronger in C3 plants than in C4 plants (Fig. S2).Additionally, plants from poorly drained wetlands (e.g., those from Mandena with MAP of ~1200 mm/yr) tend to be relatively depleted in 15 N. Also, given that members of Fabaceae are frequently associated with N-fixing bacteria (tending to lower foliar δ 15 N values) we expected that removing specimens from this family (n=378, spanning the full aridity gradient) from analysis would strengthen the observed relationship between δ 15 N and MAP.However, we found the adjusted r 2 of 0.68 unchanged, which may follow from the diverse taxa represented in this family and a range of mycorrhizal associations spread across other families. 15N values according to plant photosynthetic pathway.Plant data are separated and color coded according to plant photosynthetic pathway.Points and whiskers mark the mean and standard deviation, respectively, of plant δ 15 N values at each site.MAP data are taken from WorldClim 2.1 [26].

Figure S2. Observed relationships between MAP and terrestrial plant δ
We used BMSC, a Bayesian regression model selection algorithm [27], to further explore the effects of two continuous predictors (MAP & Coast_Distance) and two categorical factors (Plant_Pathway & Material) on plant δ 15 N values.This modeled dataset includes 2,855 δ 15 N measurements from terrestrial plants with known photosynthetic pathway, specified plant part, and collection site known within 50 km.The associated model file can be accessed on Pandora (https://pandoradata.earth/dataset/isomad-modern-biological-material) and viewed with BMSC.The Bayesian Information Criterion suggests that the most parsimonious model includes all four of the aforementioned predictors (BIC=7193.14).As expected, the model suggests that plants both closer to the coast and those from relatively arid locations have tissues relatively enriched in 15 N. Model parameters also indicate that roots are typically enriched in 15 N relative to stems and flowers and that C3 plant tissues tend to be enriched in 15 N relative to those of C4 plants.